Metal complexed serine protease inhibitors

ABSTRACT

Serine protease inhibitors are provided comprising compounds having a P site binding moiety and a divalent cation(s) chelating moiety which are pre-prepared as divalent cation(s) complexes or combined with the serine protease in the presence of divalent cation(s). The compounds are shown to have high inhibitory activity when complexed to divalent cation(s) and find use in various processes associated with serine protease isolation and inhibition.

TECHNICAL FIELD

The field of this invention is serine protease inhibitors.

BACKGROUND

Serine proteases are universally found in living organisms, fulfilling awide variety of functions. In many cases, the serine proteases havespecific targets, where precursors are activated to biologicallycompetent products to discharge their particular function. In othercases, there is a more generalized activity, where particular dipeptidesequences within larger proteins are subject to scission in the processof degradation.

Among important serine proteases are trypsin-like enzymes, such astrypsin, tryptase, thrombin, kallikrein, and factor Xa. The serineprotease targets are associated with processes such as blood clotting,complement mediated lysis, the immune response, glomerulonephritis, painsensing, inflammation, pancreatitis, cancer, regulating fertilization,bacterial infection and viral maturation. By inhibiting serine proteaseswhich have high specificity for a particular target, one can inhibit invivo numerous biological processes, which may have adverse effects on ahost.

The serine proteases have a highly conserved active site, where specificamino acids which catalyze the bond scission have a nearly identicalspatial arrangement. A complementary binding site adjacent to the activesite provides for the primary specificity of the individual serineprotease. A succession of indentations or "pockets" along the surface ofthe protease serve to bind successive side chains along the substratepolypeptide chain on either side of the peptide bond to be cleaved.These are described as P3, P2, P1 prior to the susceptible peptide bond,and P1', P2', P3' after the scissile bond in the substrate, when thepeptide sequence is represented in the customary N-to-C terminal manner.Therefore, by providing recognition elements similar to specific aminoacid side chains found in the substrate peptide sequences having highaffinity to the pockets, one can direct moieties capable of occupyingthe pocket of the active site to specific serine proteases, so as torestrict the pathways which are inhibited. There is, therefore,substantial interest in identifying inhibitors for the catalytic activesite of serine proteases which may be joined to moieties providingspecificity for a particular protease.

RELEVANT LITERATURE

Tidwell et al., J. Med. Chem. (1978) 21:613-623; Geratz et al., Arch.Biochem. Biophys. (1979) 197:551-559; and U.S. Pat. No. 4,940,723describe bis-(5-amidino-2-benzimidazoylyl)methane and analogs thereof asserine protease inhibitors. Higaki et al., Biochemistry (1990)29:8582-8586; Kang, Korean J. of Pharmacology (1988) 24:111-123; andKelly et al., Biochem. J. (1981) 199:129-136 report instances of serineproteases involving the use of zinc.

SUMMARY OF THE INVENTION

Methods and compositions are provided for the modulation of serineprotease activity. Compounds are provided, which in combination withdivalent metal cations, most preferably zinc or cobalt, or as the metalcation complexes, are used to inhibit one or more serine proteases. Thecompounds comprise a P1 binding moiety and a metal chelating moietyjoined by a linking group and spaced apart to fit in the active site ofthe target serine protease. The compounds find use in the inhibition ofserine proteases in vitro and in vivo, in assays and in the protectionof peptides and proteins from proteolytic degradation. Moreover, sincezinc is one of the more common divalent metal ions present inphysiological tissues and fluids, usually at 10-100 μM concentration,the most preferred embodiment of this invention includes zinc as thedivalent metal cation.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compounds used therein are provided for inhibition of serineproteases, particularly trypsin-like serine proteases. These enzymes maydiffer as to sequence depending upon the particular source, whetherprokaryotic or eukaryotic, the tissue from an animal source, mutants,and the like. Inhibition is achieved by contacting the target serineprotease with the inhibiting compound in the presence of aphysiologically acceptable metal ion mediator, particularly divalentmetal ion, more particularly zinc or cobalt, particularly added metalion for known inhibitors, or as a preformed complex. The inhibitor ischaracterized by having two heteroatoms which are able to complex themetal ion by having unshared pairs of electrons. Inhibition employingknown inhibitors of this structural class can be substantially enhancedby providing pre-formed complexes with the metal ion or adding the metalion to the medium in sufficient amount to provide the enhancedinhibition. Alternatively, in vitro or in vivo, where the minimum amountof zinc is present, the inhibitor need only be added to the targetserine protease to provide for the enhanced inhibitory activityresulting from the zinc complex.

Since zinc is the preferred metal ion and is present in physiologicalsamples, hereafter zinc will be used as exemplary of metal ionsgenerally, particularly exemplary of cobalt.

The zinc complexes of this invention may be characterized by thefollowing formula:

    {(B.sub.P).sub.r -(I)}.sub.n (Y).sub.q --Zn.sup.2+

wherein:

B_(P) intends the binding moiety for binding to one or more of the Psites, particularly at least P1;

I is a moiety that comprises at least one heteroatom, which is oxygen,sulfur or nitrogen and when n is 1, comprises at least two heteroatoms,spaced apart so as to be able to chelate zinc, or when n=2, has at leastone heteroatom, so that the two groups joined by Y are capable ofchelating zinc in a bidentate manner, thus providing two of the fourcoordinating ligands around zinc;

Y is a bond or linking group of not more than six, usually not more thanthree atoms in the chain, particularly carbon, which provides that thespacing when the two Is are bound to Y provides chelation of zinc; r is0 to 1, wherein the sum total of r's is at least 1;

n is one to two, and q is zero when n is one and one when n is two.

When n is 2, illustrative compounds will preferably come within thefollowing formula:

    (B.sub.P).sub.r' {α-(A)-β}

wherein:

α is a heterocycle having at least one heteroatom, which is N, O or S,or is a cyclic aromatic ring with one of these heteroatoms directlyattached, is of at least 2 carbon atoms and not more than 36 carbonatoms, usually not more than 18 carbon atoms, preferably not more than12 carbon atoms, may be mono- or polycyclic, fused or non-fused, wherethe individual rings will usually be of from 4 to 7, more usually 5 to 6annular members, may include aliphatic unsaturation, wherein aheteroatom is within 3 atoms, usually within 2 atoms, and preferablywithin 1 atom of A; there being from about 1 to 4, usually 1 to 3 andpreferably 1 to 2 annular heteroatoms, generally not more than 8heteroatoms, more usually not more than 6 heteroatoms total;

A is a bond or a linking group of from 1 to 2 atoms, usually 1 atom,which are C, N, O or S, where any remaining valences are satisfied by H,alkyl of from 1 to 2 carbon atoms, oxo, oxy of from 0 to 2 carbon atoms,halo, amino of from 0 to 2 carbon atoms, and thiol, wherein heteroatomsare bonded only to carbon, hydrogen and oxygen, particularly carbon andhydrogen;

β comprises a heterosubstituted carbon ring bonded directly orindirectly to A, where the heteroatom is N, O or S; β may be the same ordifferent from α, is of from about 1 to 36, usually 2 to 30, moreusually 3 to 18 carbon atoms, may be aliphatic, alicyclic, aromatic,heterocyclic, or combinations thereof, the number of heteroatoms comingwithin the definition for α when a heterocycle, otherwise having fromabout 1 to 6, usually 1 to 4 heteroatoms as defined above, and whereinthe heteroatom bonded to the carbon is not more than 6 atoms, usuallynot more than 4 atoms from the heteroatom of α within 3 atoms of A; βmay include aliphatic unsaturation, ethylenic or acetylenic, aromaticunsaturation, or be saturated; and wherein the two related heteroatomsare positioned to be able to occupy the coordinate complex (dsp²)chelating sites of zinc;

r' is 1 or 2;

B_(P) is the moiety binding to the P site of the serine protease and isbound to α and/or β in appropriate spatial relationship for binding tothe serine protease, usually from about 1 to 6, more usually from about2 to 4 atoms away from the heteroatoms of α and/or β in positionalrelationship to A.

α and/or β heterocycles of interest preferably have at least one sp²nitrogen atom, include 5 membered rings, such as pyrazole, imidazole,triazole, oxazole, thiazole, isoxazole, etc.; benzo-fused derivativesthereof, such as benzimidazole, etc.; six membered rings, such as pyran,pyridine, pyridazine, pyrimidine, pyrazine, dioxane, etc.; benzderivatives thereof, such as quinoline, isoquinoline, cinnoline, etc.;non-fused rings, such as 2,2'-bisimidazole, 2,2'-bis-pyridine, etc.Substituents may include halogen, oxy, amino, cyano, non-oxo-carbonyl,alkyl, or any other common substituent, preferably electron-donating,which does not sterically preclude binding or complexation stepsnecessary for the inhibitor to function.

When other than a heterocycle, β can be methoxymethyl, aminomethyl,methylaminomethyl, guanidinyl, anilinomethyl, 2,3-diaminopropyl,2-amino-3-hydroxypropyl, 2-amino-2-trifluoromethylethyl, 2-hydroxyethyl,substituted aromatics like 2-mercaptophenyl, 2-hydroxyphenyl,2-aminophenyl, 2-carboxyphenyl and substituted analogs thereof,methylacetoxy, glycinamidomethyl, cyclohexylaminomethyl, and the like.

The subject compounds may have one or more heteroatom containingsubstituents other than the heteroatoms involved in chelating. Thesubstituents will usually be not more than 6 carbon atoms, more usuallynot more than 3 carbon atoms and may include amino of from 0 to 6 carbonatoms, non-oxo-carbonyl of from 1 to 6 carbon atoms, particularly salts,esters and amides, and the sulfur and nitrogen analogs thereof, oxy oralkyloxy of from 0 to 6 carbon atoms, or aryloxy, halo, cyano, nitro,oxo, etc.

B_(P) can be guanidine, amidine, aminomethyl, amino higher alkyl,α-aminocarboxymethyl, α-aminocarboxamidemethyl, etc.

Compounds of particular interest include 5-substituted2,2'-bis-benzimidazoles, where the 5-substituent is any basic groupbonded to the basic heteroatom or group through a carbon, where thesubstituent will usually be not more than 10 atoms other than hydrogen,more usually not more than 6 atoms other than hydrogen, generallycomposed of carbon, hydrogen, nitrogen and oxygen. The other imidazolering may also be substituted with alkyl, non-oxo carbonyl, amino,aminoalkyl, amidinyl, or the like, generally of not more than 12 atomsother than hydrogen, more usually not more than 6 atoms other thanhydrogen.

When n is one, compounds of particular interest will have the followingformula: ##STR1## wherein: B_(P) has been defined previously;

A is a bond or linking group having one to four atoms in the chain,normally carbon or heteroatom, more usually one to two atoms, where theheteroatoms are particularly oxygen, sulfur or nitrogen; the linkinggroup may be substituted by other atoms, particularly to maintain thechelating heteroatoms in position to chelate with zinc; A may bealiphatic, aromatic, alicyclic, heterocyclic or combinations thereof,and may be taken together with the heteroatoms to which it is attachedto form a ring, wherein the broken line indicates that the heteroatomsmay be part of a heterocyclic ring; and

X_(a) and X_(b) are oxygen, sulfur and nitrogen, particularly sp²-hybridized nitrogen, spaced apart so as to be capable of chelatingzinc, bonded to carbon, hydrogen and oxygen, particularly carbon andhydrogen; and may be annular heteroatoms or substituted directly orindirectly to an annular atom. Of particular interest are compoundswhere the heteroatom is a heteroannular atom of a ring of from 4 to 7,preferably from 5 to 6 heteroannular members, there usually being atleast 2, more usually at least 3 carbon atoms in the ring, the remainingatoms being the heteroatoms mentioned heretofore.

The compounds may be aliphatic compounds, where the heteroatoms areflexible or held in spatial relationship by the presence of unsaturationin the linking group, e.g. ethylenic unsaturation, or being held inposition by being bonded, either directly or through a carbon atom to aring of from 3 to 7 members; the heteroatoms and linking group may bepart of a heterocyclic ring, such as in benzimidazole or pyrimidine. Thesignificant factor is that the required chelating atoms may be membersof different non-fused rings.

The heteroatoms for chelating will generally be separated by at least 2other atoms and not more than 4 other atoms, usually not more than 3other atoms, the other atoms usually being carbon. The atoms may benon-annular, annular, or combinations thereof. Of particular interest iswhere nitrogen is a heteroannular member of a 5 to 6 membered ring,where the ring has 2 nitrogen atoms separated by a carbon atom,particularly where there is a double bond between the intervening carbonatom and a nitrogen atom. Also of particular interest, are rings, asdescribed above, joined together by a bond of from 1 to 2 atoms,particularly 1 atom, where such linking group may be a carbon atom,substituted carbon atom, or heteroatom, particularly nitrogen, oxygenand sulfur. Desirably, the linking group is alpha to a nitrogen atom,more particularly where there are 2 nitrogen atoms separated by anintervening carbon atom, and bonded to the intervening carbon atom.

The binding portion of the molecule which fits into the P site,particularly the P1 site, may be an amino acid, particularly arginine orlysine, may be an oligopeptide which is the specific target site for thetarget serine protease, may be preferably amidine, amino, guanidino, orother basic moiety for trypsin-like serine protease, carboxylates orlipophilic groups for chymotrypsin-like proteases.

For the most part, the compounds of this invention will be fewer than 60carbon atoms, more usually fewer than 36 carbon atoms, particularlyfewer than 18 carbon atoms, more particularly fewer than 12 carbonatoms, having fewer than 20 heteroatoms, frequently fewer than 16heteroatoms, generally fewer than about 12 heteroatoms, particularly notmore than about 8 heteroatoms, frequently not more than about 7heteroatoms. Particularly, where the binding moiety is other thanoligopeptide, the number of heteroatoms in the molecule will be not morethan 12, more usually not more than 8, generally there being at least 2heteroatoms, more usually at least 4 heteroatoms.

Where 2 rings are joined by a linking group to form the chelatingmoiety, the linking group may be methylene, methene, carbonyl, amino,oxy, thio, isopropylidene, etc.

Instead of having 2 entities joined together by a linking group, one mayprovide for a single entity particularly fused polycyclics, havingheteroatoms at appropriate positions. Compounds of interest include1,8-diaminonaphthalene, 8-cinnoline, 5,6-diaminophenanthrene,1,8-dihydroxynaphthalene, and the like, where the positioning of theheteroatoms for chelation corresponds to the definitions described abovefor the heterocycle.

Compounds of interest include, but are not limited to:

5,5'-bis(amidinobenzimidazole)methane (BABIM);

5,5'-bis(amidinobenzimidazole)carbonyl;

2-(2'-(5'-aminomethyl)benzimidazolyl)methylbenzimidazole;

2-(2'-(5'-aminomethyl)benzimidazolyl)methyl-5-methylbenzimidazole;

2-(2'-(5'-amidino)benzimidazolyl)methylbenzimidazole;

2-(2'-benzimidazolyl)methylbenzimidazole;

2-(2'-(5'-guanidino)benzimidazolyl)methylbenzimidazole;

2-(2'-(5'-carboxy)benzimidazolyl)methylbenzimidazole;

2-(2'-(5'-amidino)benzimidazolyl)methylimidazole;

4-(2'-(5'-amidino)benzimidazolyl)methylimidazole;

2-(2'-(5'-amidino)benzimidazolyl)pyridine;

2-(2'-(5'-amidino)benzimidazolyl)methylpyridine;

1-(2'-(5'-amidino)benzimidazolyl)isoquinoline;

2-(2'-(5'-amidino)benzimidazolyl)quinoline;

3-(2'-(5'-amidino)benzimidazolyl)isoquinoline;

8-(2'-(5'-amidino)benzimidazolyl)quinoline;

5-amidino-(2'-hydroxyphenyl)benzimidazole;

5-amidino-(2'-mercaptophenyl)benzimidazole; and

5-amidino-(2'-aminophenyl)benzimidazole.

The subject compounds can be used in conjunction with zinc to inhibitserine protease enzymes, where one compound may be preferred inconjunction with a particular serine protease.

The subject compounds can be prepared in accordance with known syntheticprocedures. See, for example, Tidwell, et al., J. Med. Chem. (1978)21:613-623; and general methods for the synthesis of substituted and/orfused heterocyclic systems and their isomers, as described in"Comprehensive Heterocyclic Chemistry", Pergamon Press:Oxford, 1988.

The subject compounds may be prepared as crude mixtures comprising atleast about 50 weight %, usually at least about 90 weight % of thecomposition. Preferably, the subject compounds will be at least about 99weight % of the composition. Similarly, the zinc complexes may beprepared and have the same purity ranges. Also, when using the subjectcompositions, particularly as the zinc complex, the composition may becomplexed with a target serine protease. This zinc complex may be formedin relation to inhibiting the serine protease, as an inhibited enzymecomposition which may be activated upon removal of the zinc from theserine protease complex with a chelating agent, or the like. The serineprotease-inhibitor composition may also come within the same purityranges as indicated above for the inhibitor.

The zinc complex may be pre-prepared and isolated by combining thesubject compound with zinc at a concentration of at least about 0.01 μM,usually at least about 0.1 μM, more usually 5 μM, preferably at leastabout 50 μM or higher, depending upon the particular compound and itsK_(i). Usually, the zinc concentration will be less than about 100 mM,more usually less than about 50 mM, and generally more than about 1 μM.Generally, the amount of zinc which is added to a preparation or mediumcontaining the inhibitor will be sufficient to increase the zincconcentration by at least about 0.1 nM, more usually at least about 1μM, generally at least about 50 μM, and usually not more than about 1mM. Conveniently, the zinc concentration employed will provide that atleast about 80%, preferably at least about 90%, and more preferablysubstantially all of the inhibitor will be chelated with zinc. Inphysiological systems, the amount of zinc present will normally besufficient to provide the zinc complex, although pre-preparation of thezinc complex may enhance the inhibitory efficiency of the subjectcompounds.

Compounds sufficiently satisfying the structural criteria describedabove may be determined by use of an in vitro assay system whichmeasures the potentiation of inhibition of any serine protease by acompound of the present invention in both the presence and absence ofphysiologically relevant concentrations of zinc. Standard assay formatsfor measurement of specific serine protease activity utilize the enzymeof choice and short peptide substrates whose cleavage can bequantitated, usually by simple colorimetric methods. This assay systemcan be configured in three formats to detect if a compound satisfies thestructural requirements for inhibition potentiation in the presence ofzinc. The assay formats are:

(a) determination of the inhibition level (indicated by Ki (Baseline))without regard to adjustment of zinc levels (i.e., ambient levelspresent in reagents and distilled water);

(b) determination of the Ki (Zinc removed) in the enforced absence ofthe zinc by addition of an excess of EDTA, phenanthroline, or any othercommon cation sequestering agent which has no effect on the proteaseactivity on its own;

(c) determination of the Ki (Zinc added) in the presence of the addedzinc to adjust the assay concentration into a physiologically relevantconcentration range; animal plasma, including human plasma, can also beutilized in the assay medium to provide physiologically relevant levelsof zinc.

A compound successfully fulfills the structural requirements describedabove only if the value of the Ki (Zinc removed)>>Ki (Zinc added),indicating inhibition is potentiated (i.e., the compound is a morepotent inhibitor) in the presence of zinc. The assay formats describedabove are given as Examples below.

The assay system developed to explain the enhanced activity of acompound of the current invention in the presence of added zinc can thusbe used to discover new serine protease inhibitors of enhanced potency,in which a P1 serine protease recognition element (itself a potentialweak inhibitor) and the bidentate chelator derived from electron-richheteroaromatic functionalities have been correctly incorporated into anew composition. The compounds of the current invention are a new classof serine protease inhibitors which are composed of the following twoelements: (a) a chemical functional group which occupies the P1 site onthe target serine protease, which alone could be a serine proteaseinhibitor; and (b) a structurally adjacent bidentate chelator whichcaptures a divalent cation, such as zinc, into a complex involving His57and Ser195 of the catalytic site of the enzyme. Combination of thesedual features into a single composition provides effective serineprotease inhibition at physiological levels of divalent cations likezinc.

The component chemical functional moieties necessary for the zincpotentiation of inhibition can also be tested separately to show thatefficient and potentiated inhibition of serine proteases is onlyobserved when the proper combination of these moieties into one compoundhas been accomplished. For example, for the compound2-(2'-(5'-amidino)benzimidazolyl) pyridine, the component functionalmoieties can be formulated as benzamidine as the P1 recognition elementand 2-(2'-pyridyl)benzimidazole, a known, commercially available zincsequestering agent, as the chelating moiety. In vitro assays using thethree systems described above clearly show that benzamidine is a weakinhibitor of the serine protease trypsin, and that2-(2'-pyridyl)benzimidazole shows no inhibition activity. However, theproper structural combination of these chemical functional groups into asingle compound, 2-(2'-5-amidino)benzimidazolyl) pyridine, clearly showsthat a significant potentiation of serine protease inhibition has beenaccomplished when the compound is evaluated in the presence ofphysiological levels of zinc. Other Examples of the application of thedesign requirements for compounds of the current invention are givenbelow.

Finally, X-ray crystallography of the inhibitor-zinc-serine proteasecomplexes can be obtained by one skilled in the art using contemporarybiophysical methodologies and commercial instrumentation. Suchcrystallographic data can be used to conclusively determine if acomposition of the current invention has embodied the structuralrequirements necessary for zinc potentiation of serine proteaseinhibition. An example of such an X-ray crystallographic determinationis presented below.

Serine protease enzymes of interest include, but are not limied to,trypsin-like enzymes, such as trypsin, kallikrein, plasmin, thrombin,and tryptase; chymotrypsin-like enzymes, including chymotrypsin,cathepsin G, and chymase; elastase-like enzymes, including neutrophilelastase and elastase; and carboxypeptidase-like enzymes. These enzymesplay a role in apoptosis, blood pressure regulation, cancer,cardiovascular function, blood clotting, lysis, chemotaxis, development,digestion, fertilization, hormone processing, immune response,complement, infection: bacterial, viral, and parasitic; inflammation,mast cells, and other cells, neurologic, pain and protein secretion.Serine protease targets in medicine include for cardiovasculartreatments: thrombin, factor Xa, factor VIIa, and chymase; forinfectious diseases involving parasites, viruses and bacteria, serineproteases specific for the pathogen; for bleeding, urokinase, and tPA;for inflammation, tryptase, chymase, neutrophil elastase, andkallikrein; and for neurobiology, serine proteases associated withAlzheimer's disease, to name only a few of the available targets.

The subject compositions may be used in a variety of ways, in vitro andin vivo. By linking the subject compositions at a site which does notinterfere with their binding to serine proteases, the subjectcompositions may be used in affinity columns to isolate serineproteases. Particularly, since the subject compounds vary as to theiraffinity to specific proteases, the subject compositions can beselective in the isolation and purification of serine proteases.

Conventional techniques may be employed for linking the variouscompounds to supports, beads, macromolecules, and the like. Linkinggroups may include carboxyl groups, amino groups, thio groups, activatedolefins, or the like. The linking group may be bonded to the inhibitionmoiety or the binding moiety. Surfaces of columns or capillaries may beemployed as the affinity column or the column may be packed with avariety of beads, such as Sephadex, sepharose, latex beads, or the like.The particular manner in which the column is prepared is not critical tothis invention. In addition, the columns may be used in assays fordetecting the various serine proteases, by providing a binding profilewhich can be developed for the serine protease of interest.

The subject compositions may be also used in conjunction with substratesfor a specific serine protease, where one wishes to maintain theintegrity of the substrate in the presence of the serine protease. Thus,if one wishes to inhibit clotting as a result of thrombin activation offibrinogen, one can employ one of the subject compounds in conjunctionwith zinc to prevent the activation of the clotting cascade, retainingthe various members of the clotting cascade as precursors. In thismanner, one can monitor the amount of fibrinogen in a blood sample andwhen one wishes to initiate the clotting cascade, add chelating agentsto remove the zinc from involvement in the inhibitor complex with theserine proteases present in the sample.

One may also use physiologically acceptable serine protease inhibitorsfor the treatment of various indications. As described in U.S. Pat. No.4,940,723, serine protease inhibition can be used for the treatment ofclinical arthritis, synovitis and associated pathologies. By comparingthe activity of the subject compositions with BABIM, the compounddescribed in the '723 patent, one can employ relevant concentrations ofthe subject compositions. For example, the amount of BABIM employed withrats was 10 mg/kg of body weight and the concentration of the subjectcompounds would be adjusted in accordance with their relative activityin relation to BABIM. One can relate the Ki of the subject compound withthe Ki of BABIM to determine an initial concentration for use, followedby optimization in accordance with known ways.

The subject compositions also permit investigation of the physiologicalprocesses associated with a wide variety of biological events involvingserine proteases. The convenience of being able to inhibit serineproteases in the presence of zinc, while removing the inhibition in theabsence of zinc, permits serine proteases to be turned on and off. Thus,one has the opportunity to investigate numerous physiological processes,in the presence and absence of active serine proteases. In addition, thesubject compositions can be used in competition with other compounds,which may be antagonists, so as to be able to screen compounds havinginhibitory activity.

For use in vivo, the subject compositions will be formulated as zinccomplexes in appropriate physiological media, e.g., water, saline,phosphate buffered saline, vegetable oil, aqueous ethanol, or the like,except in those limiting cases in which the formulation components,buffers, excipients or solvent or solvent combination would causeprecipitation of the zinc complex. Usually, however, solubility andcomplex formation are well maintained at or around physiological pHvalues. Various excipients may be included as is known in the art. Theconcentration of the particular compound will vary widely, dependingupon the manner of administration, the indication for which it is beingused, the frequency of administration, the activity of the compoundbeing employed, and the like. Therefore, for each compound and eachindication, the formulation will be optimized for the particularpurpose. Generally, the inhibitor will be present in at least about 0.01weight % and not more than about 50 weight %, usually not more thanabout 5 weight %.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

The assay for the determination of Ki of trypsin was performed asfollows:

Baseline Assay Format

Two 96-well microtiter plates are used for the enzyme assays. Enzyme andsubstrate are added to the assay plate and inhibitor concentration isvaried in a dilution plate (a single dilution plate is used for 2 assayplates). The candidate compound (˜1 mg) is dissolved in DMSO (200 μl) ordistilled water (1.0 ml) and the stock concentration is calculated. Thestock solution is diluted 1:10 into the first well of a 96-wellmicrotiter plate (Falcon, cat#3918) (20 μl) and 180 μl of buffer added(in the first dilution, with the candidate compound dissolved in DMSO,the buffer is 50 mM Tris, pH 8.2, 100 mM NaCl, 0.05% Tween-20, whilewhen dissolved in water, the buffer includes 10% DMSO). Seven serial3-fold dilutions down a column of the plate (60 μl of the sample and 120μl of buffer with DMSO) are carried out and the pH is confirmed to 8.0.50 μl of sample from each well of the dilution plate is transferred to acorresponding well on the assay plate, followed by the addition of 25 μlof enzyme solution (Stock solution is diluted 1:160 in buffer with DMSOto provide a final trypsin concentration of 3.4 nM).

The assay plate is then wrapped in plastic wrap and the inhibitor (20 mMpentamidine in DMSO or candidate compound) incubated at room temperaturefor 1 hr. At the end of the incubation, 25 μl of substrate(N-tosyl-gly-pro-lys-pNA; Sigma #T6140, 0.5 mM in water) is added toprovide a total volume of 100 μl. The plate is then inserted into aplate reader and the change in absorbance at 405 nm measured for fiveminutes.

Assay Format with Zinc Removed

The assay protocol for determination of inhibition in the enforcedabsence of zinc is identical to the above Baseline assay format, withthe exception that the buffer mixture listed above is substituted byphosphate buffered saline, 0.05% Tween 20, 10% DMSO and 1 mM EDTA,adjusted to pH 8.0.

Assay Format with Zinc Added

The assay protocol for determination of inhibition in the enforcedpresence of zinc is identical to the above Baseline assay format, withthe exception that the buffer mixture listed above is substituted byphosphate buffered saline, 0.05% Tween 20, 10% DMSO adjusted to pH 8.0.To this mixture is added 150 μM zinc chloride in the form of a stocksolution of 100 mM concentration in water.

The candidate inhibitors were prepared based on the use of amidine asthe P1 binding moiety and various nitrogen based chelating agents as theinhibiting moiety. Using the assay described above, the Ki for a numberof these compounds was determined for trypsin inhibition. The followingtable indicates the results.

                  TABLE 1                                                         ______________________________________                                        Serine Protease Inhibition Data                                                              Trypsin inhibition, (Ki, μM)                                Compound         Baseline Zn removed                                                                              Zn added                                  ______________________________________                                        BABIM            0.096    88.2      0.35                                      5-5'-bis(amidinobenzimidazole)-                                                                   50       35     0.47                                      carbonyl                                                                      benzamidine         325      266    273                                       2,2'-bis-imidazole                                                                             >1000    >1000     474                                       2-(2'-(5'-amidino)benzimidazolyl)-                                                             0.019       182     0.023                                    methylbenzimidazole                                                           benzylamine      >1000       344    354                                       2-(2'-(5'-amidino)benzimidazolyl)-                                                             >1000    >1000     32.8                                      methylimidazole                                                               2-(2'-(5'-aminomethyl)benzimi-                                                                    808   >1000     1.47                                      dazolyl)methyl-5-methylbenzi-                                                 midazole                                                                      2-(2'-(5'-aminomethyl)benzimi-                                                                 1.26     >1000     1.93                                      dazolyl)methyl-5-carboxybenzi-                                                midazole                                                                      2-(2'-pyridyl)-benzimidazole                                                                   >1000    >1000     >1000                                     2-(2'-(5'-amidino)benzimidazolyl)-                                                                748      101    20.9                                      pyridine                                                                      ______________________________________                                    

X-Ray Crystallallography of an Inhibitor-Zinc Complex Bound to Trypsin

Crystals of benzamidine-trypsin were grown using MgSO₄ as theprecipitant in 100 mM Tris, pH 8.2 by the batch method of M. Krieger, L.M. Kay and R. Stroud, J. Mol. Biol. 83:209-230 (1974). BABIM-trypsincould not be crystallized de novo, nor with seeding withbenzamidine-trypsin crystals. Initial attempts to soak BABIM into thesecrystals at pH 8.2 were unsuccessful because of limited solubility andstability of BABIM at this pH in MgSO₄ -containing synthetic motherliquor. Moreover, in Tris buffer alone at pH 8.2 BABIM turns yellowwithin an hour, and after many hours begins to precipitate. In MESbuffer at pH 5.9 this transformation does not occur after several weeks.The solubility of BABIM freshly dissolved in synthetic mother liquorcontaining 415 mg/ml MgSO₄, 100 mM Tris, pH 8.2 or 415 mg/ml MgSO₄, 100mM MES, pH 5.9 was determined to be ˜100 mM.

Crystals of BABIM-trypsin (pH 5.9) were first prepared successfully bysoaking benzamidine-trypsin crystals in freshly dissolved BABIM insynthetic mother liquor at saturation at pH 5.9. The soaks were replacedwith fresh ones 4 times, about once a day. Crystals of BABIM-trypsin (pH8.2) were successfully prepared by soaking benzamidine-trypsin crystalsin freshly dissolved BABIM in synthetic mother liquor at saturation atpH 8.2 containing 0.1 mM ZnSO₄. The soaks were replaced with fresh ones5 times, about once a day. Significant amounts of yellow precipitateformed in the soaking solutions after a day.

Individual I_(hkl) data were collected for benzamidine-trypsin (pH 8.2),BABIM-trypsin (pH 5.9) and Zn⁺² -BABIM-trypsin (pH 8.2) on a SiemensMultiwire Area Detector or on an image plate detector (R-axis-II (RigakuCorporation)). Data were extracted using the XDS programs of Kabsch, orthe software provided by the Macromolecular Structure Corporation,(Houston Tex.), and an indexing program. Data collection and refinementstatistics are listed in Table 2.

For the refinement of benzamidine-trypsin, the highly refinedMIP-trypsin structure served as a template. Water molecules outside theactive site region in the MIP X-ray and neutron structures were includedin the initial phasing model. The structure was refined usingalternating cycles of (|Fo|-|Fc|), α_(c), and (2|Fo|-|Fc|), α_(c) anddifference maps (J. L. Chambers & R. Stroud, Acta Cryst. B35:1861-1875(1979), and automated least squares refinement with XPLOR (Brunger).Difference Fourier maps were computed between benzamidine-trypsin (pH8.2) and BABIM-trypsin (pH 5.9) or Zn⁺² -BABIM-trypsin (pH 8.2) to yieldinitial structures which were then likewise refined.

The (2|Fo|-|Fc|), α_(c) difference map for BABIM-trypsin at pH 5.9clearly shows one amidinobenzimidazole group of BABIM occupying the P1pocket. The benzamidine portion of it closely overlays that forbenzamidine in the benzamidine-trypsin structure. At pH 8.2, the(|Fo|-|Fc|), α_(c) and (2|Fo|-|Fc|), α_(c) maps for the Zn⁺²-BABIM-trypsin complex clearly reveal the position and orientation ofBABIM as well as a strong peak (13σ in the (|Fo|-|Fc|), α_(c) map) whichcorresponds to the position of the Zn⁺² ion coordinating two of theimidazole nitrogens of BABIM, the imidazole of His57 and the O.sub.γatom of Ser195. Thus the nanomolar binding constant of BABIM is achievedwith the synergy of Zn⁺² at concentrations of this metal at lower than100 nM. Bond distances involving the Zn⁺² ion, are similar to thoseobserved in other Zn⁺² -containing proteins. The average of 5ligand-Zn⁺² -ligand angles is 114(11)°, but the 6^(th) angle, involvingthe BABIM ligand alone (N3-Zn⁺² -N3') is only 81°. The BABIM moleculepivots to an altered position in the structure of trypsin-BABIM-Zn⁺² atpH 8.2, to allow the benzimidazoles of BABIM to form part of the Zn⁺²binding site. The pivoting causes an insignificant change in theposition of the amidine group in the P1 pocket, but a large shift (˜5 Å)in the position of the other amidine group. An NH group of one terminalamidine nitrogen interacts with main chain carbonyl 41 through bridginghydrogen bonds, while another NH group of the other terminal amidinenitrogen forms a direct hydrogen bond with the main chain carbonyl 244of a symmetry related molecule.

                  TABLE 2                                                         ______________________________________                                        Crystallography of Trypsin-Benzamidine, Trypsin-BABIM-SO.sub.4.sup.-2,        pH 5.9, and Trypsin-BABIM-Zn.sup.+2, pH 8.2                                           Trypsin-Benzamidine                                                                       Trypsin-   Trypsin-                                               (1 BNZ)                                                                              (2 BNZ)  Babim-SO.sub.4.sup.-2                                                                    Babim-Zn.sup.+2                            ______________________________________                                        Parameters.sup.a                                                              #Atoms (includ-                                                                         4114.sup.a                                                                             4095.sup.a                                                                             1926     1955                                     ing disorder)                                                                 #Waters (includ-                                                                        225      229      211      214                                      ing disorder)                                                                 #discretely dis-                                                                        23       18       20       21                                       ordered groups.sup.b                                                          #discretely dis-                                                                         2        5        4        7                                       ordered waters                                                                #side chains with                                                                       12       13       16       13                                       refined occs.sup.c                                                            #waters with re-                                                                        90       91        0        0                                       fined occs                                                                    Diffraction                                                                   Statistics                                                                    Resolution.sup.d (Å)                                                                8.00-1.50                                                                              7.00-1.70                                                                              8.00-1.77                                                                              8.00-2.04                                #Observations      63142                                                      #Reflections.sup.d                                                                      24173    17697    12081    8337                                     F/σ cutoff                                                                         2.4      0        0        0                                       .sup.R merge.sup.e (%)                                                                  16.1     15.4     13.4     15.8                                     .sup.R cryst.sup.f (%)                                                        Completeness.sup.c                                                                      69.2     69.4     55.9     55.6                                     (%)                                                                           rms deviations.sup.g                                                          Bond lengths (Å)                                                                     0.016    0.016     0.014    0.017                                  Bond angles (°)                                                                   3.7      3.7      2.9      3.8                                     Torsion angles (°)                                                               26.7     26.3     26.1     26.4                                     ______________________________________                                         .sup.a Restrained, isotropic temperature factors were refined for all         structures. Hydrogen atoms were included in the refinement of the             trypsinbenzamidine structures.                                                .sup.b not including waters.                                                  .sup.c Density for all side chain atoms or for terminals atoms in these       groups was weak absent and temperature factors were high. Discretely          disordered groups are not included in this category. Occupancies for          poorly defined atoms were refined.                                            .sup.d refers to refinement limits.                                           .sup.e .sup.R merge = Σ.sub.h Σ.sub.i |I(h).sub.i -      <I(h)>|/Σ.sub.h Σ.sub.i I(h).sub.i, where I(h).sub.i     is the ith observation of the intensity of reflection h.                      .sup.f .sup.R crypt = Σ(F.sub.o - F.sub.c)/ΣF.sub.o.              .sup.g Root mean square deviations from ideal bond lengths and bond           angles.                                                                  

It is evident from the above results, that by using metal ion complexes,particularly zinc, or metal ion complexes, particularly zinc, incombination with compounds comprising a P site binding moiety and ametal ion chelating moiety, which compounds can fit at the active site,extremely active serine protease inhibitors can be produced. Bymodifying the P site binding moiety, the chelating compounds can bedirected to a variety of different serine proteases with highspecificity. In this manner, one may inhibit serine protease activity invitro and in vivo, in studying physiological processes, in preventingdegradation of proteins which are specific substrates of serineproteases, in inhibiting bacteriological action, and in treating avariety of indications, where the pathology is associated with activeserine proteases.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. In a method for inhibiting a catalytic activityof a serine protease with a serine protease inhibitor in a mediumcomprising the serine protease and the inhibitor, wherein the inhibitorcomprises a compound of Formula (a):

    {(B.sub.p).sub.r -(I)}.sub.2 Y                             (a)

in which: Y is a bond or linking group of from 1 to 6 atoms; B_(p) is amoiety for binding to one or more P sites of the serine protease; r is 0or 1, with the proviso that at least one B_(p) moiety is present; and Iis a moiety that comprises at least one heteroatom selected from N, Oand S, which heteroatom is within 1 to 3 atoms of Y and 1 to 6 atoms ofany B_(p) moiety that is attached to the I moiety comprising theheteroatom, with the proviso that at least one I moiety is a fused ornon-fused, mono- or polycyclic moiety of 3 to 36 carbon atoms, whereinthe cyclic moiety is attached to Y via an imidazole ring and anyadditional rings comprising the cyclic moiety each contain 4 to 7annular members; the improvement which comprises (i) having sufficientzinc in the medium, (ii) adding sufficient zinc to the medium or (iii)providing the inhibitor to the medium as a zinc complex, such that theinhibitor, the zinc and a catalytic site of the serine protease form aternary complex whereby the catalytic activity of the serine protease isinhibited.
 2. In a method for inhibiting a serine protease with a serineprotease inhibitor comprising a bis-benzimidazole in a medium comprisingsaid serine protease and said inhibitor, wherein said inhibitorcomprises nitrogen atoms of said bis-imidazole in spatial relationshipto chelate zinc, the improvement which comprises:adding sufficient zincto said medium to have all of said inhibitor which is bound to saidserine protease as a zinc complex or providing said inhibitor as a zinccomplex.
 3. A method according to claim 2, wherein saidbis-benzimidazole inhibitor is substituted with an amidine.